Research Project:
BIOLOGICAL CONTROL OF INVASIVE AND EXOTIC PESTSTitle: Progress on resolving the Gonatocerus tuberculifemur complex: neither COI nor ITS2 sequence data alone can discriminate all the species within the complex, whereas, ISSR-PCR DNA fingerprinting can

Interpretive Summary: Accurate identification of closely related natural enemies is critical to the success of biological control programs. Improper identification has lead to failure of several programs. The aim of the current study was to resolve the Gonatocerus tuberculifemur complex. A complex refers to the fact there could be or that there are more than one species present. G. tuberculifemur is a potential candidate biological control agent for the glassy-winged sharshooter in California. We utilized two molecular methods, DNA fingerprinting and DNA sequencing of two genes (COI and ITS2) to analyze five possible species within the G. tuberculifemur complex. COI sequence variation was only able to discriminate three species out of the five, ITS2 sequence data discriminated four out of five, whereas, DNA fingerprinting discriminated all of the five species within the complex. No single gene was able to discriminate all of the species. Based on the current data, and preliminary cross-mating studies, it appears that there could be five species within the G. tuberculifemur complex. This data is very important to the biological control program in California.

Technical Abstract:
We utilized two molecular methods to aid in resolving the Gonatocerus tuberculifemur complex, potential glassy-winged sharpshooter (GWSS) biological control candidate agents from South America. The two methods used were DNA sequencing of both the mitochondrial cytochrome oxidase subunit 1 gene (COI) and the ribosomal internal trnascribed spacer region 2 (ITS2) and inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) DNA fingerprinting. COI sequence variation, as shown previously, was able to discriminate G. tuberculifemur individuals belonging to clades 1 and 2, but G. tuberculifemur specimens collected from the type locality (Pucara) or G. sp. 3. G. tuberculifemur individuals emerging from Hortensia similis (Cicadellini leafhopper) formed a new clade (Y). On the other hand, ITS2 rDNA sequence data could not discriminate G. tuberculifemur individuals belonging to clades 1 and 2. However, ITS2 was able to discriminate G. sp 3 and G. tuberculifemur specimens from the type locality (Pucara), forming two new clades, Z and X, respectively. Speciments emerging from H. similis were also discriminated by ITS2 analysis. Interestingly, the separation of all of the species or strains within the G. tuberculifemur complex was accomplished by ISSR-PCR DNA fingerprinting. No single gene (COI and ITS2) sequenced was able to discriminate all of the species within the G. tuberculifemur complex. Based on the current data, it appears that there could be five species within the G. tuberculifemur complex. Because G. tuberculifemur is under consideration as a potential biological control agent for the invasive GWSS in California, understanding possible cryptic variation of this species is critical.